A safe and effective human immunodeficiency virus type one (HIV-1) vaccine is urgently needed to stem worldwide AIDS pandemic, but still remains elusive. Among all the HIV-1 vaccine modalities developed and tested thus far, nef-deleted live-attenuated HIV-1 vaccines (nef-LAV) confer the best protection against HIV-1 acquisition in simian immunodeficiency virus/rhesus macaque model of HIV-1 infection; however, its safety concerns preclude its clinical use. We at University of Nebraska-Lincoln in the past years have developed an unnatural amino acid (UAA)-mediated blank codon suppression approach to control HIV-1 protein biosynthesis. Our approach entails the manipulation of essential protein biosynthesis through blank codon (codons that do not encode a natural proteinogenic amino acid, such as nonsense triplet codon and frameshift codon) suppression that is precisely controlled by UAA that is not found in nature. We applied this technology to HIV-1 vaccine research by introducing several sets of blank codons coding for UAA into virus genome to control HIV-1 replication. We successfully demonstrated that we could turn HIV-1 replication on and off in vitro. We hypothesize that our novel strategy to allow live-attenuated HIV-1 to replicate only in the presence of the three artificial regulatory elements that are not found in nature (novel nef-LAV) will greatly improve the safety of conventional nef-LAV while still maintains its good efficacy against HIV-1 acquisition. We therefore propose to test this hypothesis with three specific aims. Specific Aim 1, to control the replication of novel nef-LAV in vitro, of which we will apply and further optimize blank codon suppression strategy and incorporate suppression tRNA-aaRS pair into the HIV-1 genome to control HIV-1 replication by supplementing or withdrawing UAA in mammalian cells in vitro. Specific Aim 2, to control novel nef-LAV replication in vivo, of which we will compare and contrast the in vivo replication capacity and kinetics of the novel nef-LAV that has passed multi-cycle replication tests in vitro in the Specific Aim 1 with conventional nef-LAV using humanized- BLT mice. The results of the Specific Aim 2 will be used to further optimize the novel nef-LAV virus design and development in the Specific Aim 1. Specific Aim 3, novel nef-LAV in vivo protection, of which we will compare the protection rate of novel nef-LAV vs. conventional nef-LAV against HIV-1 rectal challenge using hu-BLT mice. Since our strategy can greatly improves the safety of nef-LAV by providing additional safety locks on HIV-1, it makes the modified nef-LAV practical in clinical trial. In addition, the approaches/tools that are developed and further optimized in this research can be applied to the generation of vaccines against other pathogenic viruses or bacteria, and therefore is expected to exert broader impact on vaccine research.